Autologous bone marrow cell transplantation in the treatment of HIV patients with compensated cirrhosis

Abstract Liver stem cell therapy is a promising tool to improve decompensated liver cirrhosis (DLC). Especially in patients infected with human immunodeficiency virus (HIV), the condition of the liver may be aggravated by antiretroviral therapy. A total of 21 patients diagnosed with DLC and HIV infection were divided into two groups as follows: those who received (combination therapy group, 14 patients) and those who did not receive (routine therapy group, 7 patients) bone marrow cell transplantation through the portal vein. Two patients died of surgery-related complications in the combination therapy group. The results showed that the survival rate was 85.7% in the combination therapy group after 2 years of follow-up, which was significantly higher than the 14.3% in the conventional therapy group (P<0.01). After treatment, the liver function score decreased significantly in the combination therapy group at 1 (t = 4.276, P = 0.000), 3 (t = 9.153, P = 0.000), and 12 (t = 13.536, P = 0.000) months, the levels of albumin were significantly increased, and the total bilirubin level and prothrombin time were significantly reduced or shortened as compared with the routine therapy group (P<0.05 or <0.01). The white blood cell count, hemoglobin, platelet count, and CD4+ and CD8+ levels were significantly higher in the combination therapy group at different time points as compared with the routine therapy group (P<0.05 or <0.01). In summary, the combination therapy is effective in HIV-infected patients with DLC and useful for the recovery of liver function and cellular immune function but may increase the risk of severe complications after surgery.


Introduction
Decompensated liver cirrhosis (DLC) is a stage in the progression of liver cirrhosis. It is associated with functional impairment of liver and portal hypertension. The main causes of liver cirrhosis include hepatitis B virus (HBV) [1,2], alcohol [3], hepatitis C virus (HCV) [4], and autoimmune liver disease. Stem cell therapy has generated great interest due to an increased incidence of chronic DLC and the shortage of organ donors for liver transplantation [5]. Moreover, DLC has a poor prognosis with elevated short-term mortality, even in steroid-treated patients. Thus, early liver transplantation can be an option for selected patients but is unavailable to the majority of patients [6]. Stem cell-based strategies have been tested as an alternative to organ transplantation.
Although anti-fibrotic drugs have been developed, their efficacy on liver disease is limited. The studies on bone marrow cells (BMCs) have facilitated the application of cellular therapeutics. Furthermore, hepatocytes from bone marrow (BM) have been found in mouse and human livers after BMC transplantation, suggesting that BMCs contribute to liver regeneration [7][8][9][10]. Also, the effect of BMCs injected into fibrotic or injured livers has been investigated. However, BMCs must undergo in vitro selection and culture for clinical application. Therefore, a clinical trial using mesenchymal stem cells (MSCs)

Statistical analysis
All patients were followed up for more than 2 years. Descriptive data are expressed as mean + − standard deviation (mean+ − SD), whereas enumeration data are expressed as a rate. The differences between the samples were analyzed using the rank-sum analysis. Survival analysis was used to compare the survival rates between the two groups. P-value <0.05 was considered statistically significant.

Comparison of pre-treatment baseline data between the two groups
The baseline data of the two groups are shown in Table 1. No statistical differences were detected in the age, weight, body mass index, CD4+, CD8+, prothrombin time, albumin, total bilirubin, WBCs, platelet, hemoglobin, and liver function between two groups (P>0.05).

Efficacy of combination BMC transplantation with splenectomy and follow-up
In the combination therapy group consisting of 14 patients, 2 showed intraperitoneal hemorrhage on the day of surgery. The surgical wounds in these patients oozed profusely when the operation was repeated. The bleeding wounds were treated with suture ligation and argon laser knife. Both patients died of abdominal bleeding and liver failure 2 days after the surgery. All other patients were followed up, the appetite and physical strength of the patients improved, and ascites decreased or disappeared [ Figure 1A (a,b)]. After 4 weeks, the serum biochemical indexes improved gradually and returned to normal. After 12 weeks, the Child-Pugh grade reached grade A. The HIV, HBV, and HCV viral loads were not detected. In the routine therapy group consisting of 7 patients, 6 with nonsurgical treatment died in 2 years due to gastrointestinal bleeding and liver failure, and 1 died of liver failure in the third year. As shown in Figure 1B, the survival rate was 85.7% in the combination therapy group after 2 years of follow-up, which was significantly higher than 14.3% in the conventional therapy group (P = 0.003).
As shown in Table 5 and Figure 2D, the total bilirubin levels in the liver secretion function were not significantly different between the two groups before the treatment (t = 1.612, P = 0.124). However, after the treatment, the levels decreased significantly in the combination therapy group at 1 (t = 0.013, P = 0.990), 3 (t = 0.147, P = 0.885), and    12 (t = 3.263, P = 0.007) months as compared with those before the treatment. The levels of total bilirubin in the combination therapy group were significantly lower as compared with those in the routine therapy group at 12 months (t = 3.758, P = 0.001).

Discussion
HBV and HCV infections may lead to DLC, which is a critical condition with high morbidity and mortality and without adequate treatment. However, for HIV-infected patients, the condition of the liver might aid the immune reconstitution with ART that could be otherwise damaged. Therefore, no curative treatment for HIV-infected patients with DLC leads to the death of patients from liver failure or opportunistic infection [12]. Hitherto, liver transplantation is the most effective treatment for DLC, which also has several limitations, including the shortage of organ donors and high medical expenses. Therefore, stem cell therapy is an alternative option for liver transplantation [12]. Recent evidence showed that stem cell therapy attenuated the clinical conditions of patients with cirrhosis, and improved the liver functions. Stem cells possess the ability of symmetrical self-renewal and pluripotency. Somatic stem cells harbored transgerm layer differentiation potential for migration to the damaged areas; these cells later developed into tissue-committed stem cells [13][14][15]. Numerous cytokines and growth factors are secreted by BMCs, which promote their migration into injured tissue and facilitate repair. Several studies demonstrated that a shortened telomere in the liver cells results in the failure of cell replication during the progress of liver cirrhosis. In mammals, the lost liver cells can be replaced by the remaining hepatocytes. However, sustained injury elicits hepatocyte senescence, which in turn, activates liver progenitor cells, known as oval cells. These oval cells have been reported to up-regulate BM-hematopoietic stem cells (BM-HSCs). Both hepatocytes and cholangiocytes can be derived from BM-HSCs. Cell fusion is a critical mechanism during this process. Additionally, BM-HSCs may also promote the proliferation of endogenous hepatocytes to repair liver injury [16,17]. Therefore, stem cell transplantation has been demonstrated to be effective and safe in the treatment of several liver complications, such as post-hepatic cirrhosis, autoimmune liver disease, and alcoholic liver cirrhosis. The function of the liver can be completely restored after stem cell transplantation for patients with alcoholic liver cirrhosis. However, virus-infected patients may need continuous transplantation to replace the damaged stem cells.
Recently, for autologous BMC transplantation, 100-200 ml of autologous bone marrow was needed to isolate MSCs by gradient centrifugation. Then, the isolated MSCs were injected into the liver via a hepatic artery [18] that supplies <33% of the liver blood within a short circulating time. Moreover, the hepatic portal vein is optimal for stem cell transplantation because of its long circulating time and an extended occupation in the liver. Additionally, high levels of hepatotropic cytokines and nutrients also benefit the maintenance of stem cells. Only a few side effects, such as embolism, are observed owing to the small diameter of BMCs. The present study demonstrated that the survival time in the combination therapy group was significantly longer than that in the routine therapy treatment group. Whether stem cells are effective in treating DLC is controversial. A meta-analysis showed that umbilical cord hepatocytes improve the liver function, relieve clinical symptoms, and improve the quality of life in patients with DLC as compared with the traditional supportive care [19]. Another meta-analysis found that tissue-derived stem cell treatment improves the liver function without complications; however, no difference is detected in the liver function and survival as compared with routine therapy [20]. A randomized controlled study in 2018 found that granulocyte colony-stimulating factor with or without hemopoietic stem-cell infusion did not improve liver dysfunction or fibrosis and might be associated with increased frequency of adverse events as compared with routine treatment [21]. The current study found that splenectomy combined with BMC transplantation through the portal vein not only improves the liver function but also improves the 2-year survival rate. Whether or not splenectomy is valuable for the body's immune reconstruction needs further investigation. Therefore, combination BMC transplantation with splenectomy is an effective technique for the infusion and growth of stem cells in the liver.
BM contains MSCs, HSCs, vascular progenitor cells, many precursor cells, and cytokines. Previous studies demonstrated that pulmonary embolism might occur in patients with multiple fractures due to the infiltration of BM into the venous system [22,23]. However, this embolism might be caused by the massive release of tissue factors and adipocytes from the sites of fracture. The capillary network between the hepatic portal vein and hepatic vein can eliminate components such as adipocytes, avoiding the occurrence of embolism during BMC transplantation. The present in vivo study did not detect any fat embolism in the hepatic portal venous system and the lung after the infusion of autologous BMCs at different time points. Moreover, the results demonstrated that patients who underwent autologous BMC transplantation combined with splenectomy exhibited normalized WBCs and platelet counts, elimination of ascites, and restored liver function. All patients were classified as Child-Pugh class A, suggesting that BMC infusion improve liver regeneration.
Three months after splenectomy and autologous BMC infusion, the number of CD4+ T lymphocytes was >300 cells/μl in the patients with no side effects. The liver functions were restored in 12 with no abnormality in the differential blood count. The CD4+ T lymphocytes of these patients were stabilized at >500 cells/μl. Splenectomy is known to avoid spleen-induced damage of various blood cells, leading to rapid repair of WBCs, red blood cells, and platelets. BMCs drive the localization, differentiation, and proliferation of liver-specific cells to renew the liver function [24]. However, CD4+ T lymphocytes are commonly developed in the thymus from precursor T lineage cells migrated from the BM. Then, the mature CD4+ T cells are released into peripheral blood [25]. The application of ART in HIV-infected patients helps to rebuild the immune system with an increased number of CD4+ T cells. In elderly patients, the number of CD4+ T cells increases slowly due to the involution of thymus. The present study demonstrated that the number of CD4+ T cells increased rapidly after autologous BMC transplantation in the 12 HIV-infected patients with liver cirrhosis, which was faster in the younger patients as compared with the elderly. The precursor T lineage cells derived from the infused BMC were implicated as a critical tool for treating HIV-infected patients. Therefore, splenectomy and autologous BMC infusion promote cellular immunity in patients with HIV infection and liver cirrhosis.

Conclusions
Splenectomy, combined with autologous BMC infusion through the portal vein, is beneficial to HIV-infected patients with DLC. It promotes the recovery of liver function and cellular immune function but may increase the risk of severe complications after the surgery.

Data Availability
All data generated and/or analyzed in this study are included in this manuscript.